A virulent subclone of E. coli sequence type 131 (ST131), called ST131-H30, has become the dominant extraintestinal pathogenic E. coli lineage in the US, where it kills thousands of patients each year. Multidrug resistance is a hallmark of ST131-H30-including production of the CTX-M-15 extended spectrum beta-lactamase (ESBL)-making its clinical management particularly challenging. The ability of ST131-H30 to colonize more frequently and persistently than other strains is thought to be important to its disproportional dis- ease burden. Yet, in our preliminary studies, we have observed that some people fail to become colonized with ST131-H30 despite prolonged household exposure. This strongly suggests that factors other than ST131- H30's intrinsic traits influence colonization. We know that, in the human GI tract, ST131-H30 does not live alone, but rather, as a minor component of a complex microbial community, within which it may be subjected to intense inter- and intra-species competition. In our previous microbiome studies of other body sites, we observed predictable relationships between commensal bacteria and opportunistic pathogens. This led us to hypothesize that distinct components of the GI microbiota, including bacteriocin-producing E. coli strains, may competitively exclude ST131-H30. Approach: By leveraging an ongoing longitudinal study of 600 participants, who will contribute > 7,000 fecal specimens, we will conduct two case-control studies to identify the microbiota unique to individuals who remain free of ST131-H30 for 12 months despite ongoing household exposure to ST131-H30 (elite non-colonizers), as compared to individuals with persistent colonization by ST131-H30 for 12 months (elite colonizers) and individuals who newly acquire ST131-H30 during the study (acquirers). We will identify bacterial species most predictive of resistance to ST131-H30 GI colonization using indicator species and decision tree analysis. The decision model will be validated using fecal microbiota data from an additional 300 participants from another cross-sectional survey. To explore intra-species competition, we will identify commensal E. coli strains antagonistic to ST131-H30 using in vitro inhibition assays, and will cross-validate the in vitro results by assessing the antagonistic strains' association with ST131-H30 exclusion using data from the longitudinal and cross-sectional cohorts. Innovation: Our approach is innovative in attempting to define mechanisms that influence GI colonization with ST131-H30; its focus on elite non-colonizers; its inter- and intra- species investigation of the microbiome within an ongoing longitudinal epidemiologic study; and its goal of identifying commensal bacteria antagonistic to a single virulent E. coli subclone. The expected outcome of this study is to identify commensal bacteria that can exclude ST131-H30 from the GI tract. With emerging resistance neutralizing the effectiveness our current antibiotic armamentarium, we must find new ways to prevent the spread of multidrug-resistant colonizing pathogens. These results could lead to novel, probiotic strategies for interrupting the transmission of ST131-H30 and future multidrug-resistant colonizing pathogens.